My research is aimed at developing, in depth, a method that I have invented for the mapping of the active sites of enzymes. The key is a substrate which is active site directed, and which is converted by the enzyme into a chemically reactive species. Our best substrate, to date, is an N-nitrosoamide of D-phenylalanine and the reactive species is the benzylcarbonium ion (the general approach has been termed k(cat) or suicide inhibition, or enzyme-activated-substrate inhibition). The chief advantage of my method is that the amide linkages making up the chain of the protein are alkylated by the carbonium ions on the oxygen atom (to produce imidates) and labilized in the process. In effect, each "hit" leads to chain cleavage. Further, the two peptides formed are normal, underactivated peptides which can be handled by the usual techniques of the protein chemist. We are currently engaged in working out the chemistry of this approach with the enzyme a-chymotrypsin, and we are about one fourth the way through the protein chemistsry that is involved. Amino acid analyses for tryptophan, the sequencing of peptides, and high resolution separation of peptides are important aspects. In addition to O-alkylation referred to above, the carbonium ion intermediates also alkylate N,S, and probably C during the inhibition and techniques of the protein chemist will be used to identify these sites. We also have evidence that photoaffinity labelling may proceed in part through imidate formation and we are pursuing that lead with new diazoalkyl substrates. We also plan applications to elastase, subtilisin and other enzymes. If the method lives up to its promise, it should be of considerable use to enzyme chemists, biologists utilizing photoaffinity labelling, etc. The principles involved may also be useful in the design of medicinal drugs.